The first objective is elucidation of the function of the spacer sequence within carA from Pseudomonas aeruginosa. Comparison of the derived amino acid sequence of carA with the amino terminal amino acid sequence of the small subunit of carbamoylphosphate synthetase revealed that the amino acid residues specified by four codons are not present in the polypeptide. This lack of colinearity can be explained on the basis of ribosomal hopping during translation or polypeptide splicing. In vivo and in vitro chase-labeling experiments as well as mutagenesis studies will distinguish between the two models. Studies on expression of carA in heterologous systems will assess the requirement of a transacting factor. Site-directed mutagenesis will be employed to investigate the function of the spacer sequence and to determine whether it plays a role in regulation of expression. Mutagenesis will be also employed to identify the sequence features that determine ribosomal hopping or polypeptide splicing. The results should be of general significance to our understanding of the function of spacer sequences within structural genes. The second objective is elucidation of the mechanism of regulation of carAB expression. Quantitative primer extension and mutagenesis experiments will be employed to test the hypothesis that the multiple transcripts initiated upstream of carA are differentially regulated by pyrimidines, arginine, and an alternative sigma factor. Studies with fusions and mutagenesis will test the hypothesis that regulation of carAB involves an attenuation mechanism. The gene encoding a regulatory factor involved in control of expression of certain genes of biosynthesis and aerobic catabolism of arginine will be identified by insertional mutagenesis. This gene will be cloned, characterized, and its product identified. A long term goal is to isolate the regulatory product and to study its interactions with the control region. The proposed studies are of significance in view of the limited information available on regulation of chromosomal genes in this opportunistic pathogen. Comparative but limited studies on regulation will be carried out also in P. stutzeri to determine if the absence of non-colinearity relates to a different regulatory pattern.